NO159941B - PROCEDURE FOR PREPARING AC LEUCIN DERIVATIVES. - Google Patents

Abstract

Novel compounds of the formula <IMAGE> I wherein A signifies the group <IMAGE> or -(CH2)5-, which inhibit pancreas lipase and can be used for the control or prevention of obesity and hyperlipaemia, are disclosed. The inventive compounds can be produced by the cultivation of microorganism Streptomyces toxytricini, identified as NRRL 15443.

Description

The present invention relates to a method for producing compounds with the general formula

where A is the group in aerobic fermentation of streptomyces toxytricin 85 - 43,. NRRL 15443 . Formula I comprises (2S,3S,5S,7Z,10Z)-5-[(S)-2-formamido-4-methyl-valeryloxy-2-hexyl-3-hydroxy-7,10-hexadecadieno-acid lactone of formula hereinafter denoted by lipstatin, and (2S,3S,5S)-5-C(S)-2-formamido-4-methyl-valeryloxyJ-2-hexyl-3-hydroxy-hexadecanoic acid lactone with formula

which is hereinafter referred to as tetrahydrolipstatin.

These compounds are new and have valuable pharmacological properties. In particular, they inhibit pancreatic lipase and can be used to combat or prevent obesity and hyperlipaemia.

The object of the present invention is the production of compounds with the above-mentioned formula I.

The digestion of fat (triglycerides) that is taken up with food takes place in the intestine by pancreatic lipase. Pancreatic lipase cleaves the primary ester bonds of triglycerides, with free fatty acids and 2-monoglycerides being produced as products. These products can then be resorbed and utilized. By inhibiting the pancreatic lipase, the above-mentioned breakdown of dietary fat and thus also the resorption and utilization of these substances are partly prevented; triglycerides are excreted unchanged.'

The inhibition of pancreatic lipase by means of compounds of formula I can be demonstrated experimentally, as the oleic acid released by the cleavage of triolein by porcine pancreatic lipase is measured titrimetrically. To an emulsion containing 1 mM taurodeoxycholate, 9 mM taurodeoleate, 0.1 mM cholesterol,.

1 mM egg lecithin, 15 mg/ml ESA, 2 mM tris-HCl, 100 mM sodium chloride, 1 mM calcium chloride and the substrate triolein, the compound of formula I dissolved in ethanol or dimethylsulfoxide (10% of the emulsion volume) is added and start the reaction by adding 100 µg (175 U) porcine pancreatic lipase. During the reaction, the pH is kept at 8 by adding caustic soda. IC50 is the concentration at which the lipase activity is half-maximally inhibited. The following table I shows the investigated IC5Q values for the compounds of formula I and tasks above the acute toxicity (DLj-q after single oral administration to mice) .

The inhibition of the resorption of fat taken up with nutrition,

which is effected by means of pancreatic lipase inhibition, can be shown in a double labeling experiment in mice. For this purpose, the experimental animals are given a test meal containing ^H-triolein

14

and C-oleic acid, and a compound of formula I. When measuring the radioactivity, the amount of H-trio-14 is then examined

lein and C-oleic acid (in % of the administered amount) which are excreted together with the excrement. The results which appear in subsequent table II show that, compared to untreated control animals, the excretion of unchanged triglyceride is greatly increased and the excretion of oleic acid is largely unchanged.

The experiments were carried out with a preparation containing approximately 10% lipstatin. The indicated dose is the administered amount of lipstatin.

According to the invention, the compounds of formula I can be prepared

read by that man

a) in the preparation of the compound of formula Ia aerobically growing streptomyces toxytricin 85-13 NRRL 15443 in an aqueous

culture medium contains suitable carbon and nitrogen sources and inorganic salts, and separates the produced compound of formula Ia from the culture liquid, or b) when producing the compound of formula Ib, hydrates the compound of formula Ia.

Streptomycete strains producing lipstatin, the compound of formula Ia, were isolated from bottom samples from various locations. As an example, mention should be made of a microorganism that was isolated from a bottom sample found in Mallorca, Spain, which was given the laboratory name Streptomyces sp. 85-13

and as at CBS, Baarn (Netherlands) was identified as Streptomyces toxytricini Preobrazhenskaya & Sveshnikova

(cf. Bergey's Manual of Determinative Bacteriology, 8th Edition, p. 811). It was then given the new designation Streptomyces toxytricinini 85-13. A freeze-dried sample of this strain was deposited on 14 June 1983 at the Agricultural Research Culture Collection, Peoria, Illinois, under the designation NRRL 1544 3.

In what follows, the identification of Streptomyces sp. 85-13:

Media

The composition of the media used is described in Int.J. Syst. Bacteriol. 1966, 16, 3; 313-321.

Nonomura diagram

Nonomura used the results of the International Streptomyces Project (ISP) for the classification of the Streptomyceten species (J. Ferment.Technol. 1974, 52,2).

Colors

The names and code numbers for aerial mycelium colors come from Tresner & Backus, "System of color wheels for streptomycete taxonomy". The colors of the colony backs come from H. Prauser<1>'s selection from Baumann's "Farbtonkarte Atlas I".

Methods

The ISP method was used (cf. Int.J. Syst.Bacteriol. 1966, 16, 3; 313-340).

I. Agar cultures after 16 days at 28°C (double determination)

a) Oatmeal lager

Growth: very good; colonies: thin, spreading; aerial mycelium: wing-alder-like, rose-brown (Light Brown 57); colony reverse: yellowish (Pr. Coo-3-m) with broad purplish-grey edges (Pr. Oc-6-c); soluble pigments: indistinct.

b) Starch-salt agar

Growth: good; colonies: thin, spreading; aerial mycelium: velvety, rose-brown (Light Brown 57) with white sector; colony reverse: dark straw colored (Pr. Coo (Cr)5a), edges and other zones pink (Pr. Oc-5-b) with some dark red-brown dots (Pr. 0-5-5 (r)); soluble pigments: indistinct. The starch-degrading activity is very pronounced.

c) Glycerin-asparagine agar

Growth: good; colonies: thin, spreading; aerial mycelium: velvety, light pinkish brown (R4ec: Grayish Yellowish Pink); colony reverse: orange (Pr. Oc-3-m/r); soluble pigments:

pale rose-brown.

d) Yeast-malt fog

Growth: good; colonies: thin, large; aerial mycelium: velvety,

reddish brown (4ge: Light Grayish Reddish Brown 45); colony reverse: yellow (Pr. Coo-4-5) and dark brown (Pr. Oc-5-r); soluble pigments: very pale yellow-brown.

II. Agar cultures after 62 days at 28°C (double determination)

a) Oatmeal lager

Growth: good; colonies: thin, spreading; aerial mycelium: powdery-velvety, cinnamon-colored (R-4ie: Light Brown (57)-Cork Tan) with broad, light border (R. 5gc: Light Reddish Brown (4.2)-Peach Tan); colony reverse: yellowish brown with ocher yellow (Pr. Coo-3-a) edge, light gray towards the lighter center (Pr. Oc- 4-r); soluble pigments: light ocher brown.

b) Starch- salt fog

As on oatmelager, but with a stronger grey-brown backside (Pr.

Oc-6-c) and with dark brown spots and rings at the ends of the cross-smear.

c) Glycerin-asparagine agar

As on starch-salt agar, but paler, light-beige (5ec: Grayish Yellowish Pink 32-Dusty Peads). Reverse: ocher yellow (Pr.: Coo (=Cr)-4b), lighter in the centre; no soluble pigments.

d) Yeast-malt fog

Growth: moderate; colonies: solid such as on oat agar,

but with a very narrow, pale-gray border; reverse side: dark yellow (Pr. Coo-4-b) dark brown near the edge; soluble pigments: indistinct .

III. Melanoid pigments

Peptone oat extract agar: after 24 hours negative, after

48 hours positive; Tyrosine agar: after 24 hours positive, after

48 hours positive.

IV. Morphology of the spore-forming aerial mycelium

Section: Spira-Retinaculum Apertum. Branch type: Sympo-dial. The spirals are often irregular, with up to 5 turns and different diameters.

V. Utilization of C sources

Little or only sporadic growth on arabinose, xylose, inositol, mannitol, fructose, rhamnose, sucrose, raffinose.

WE. Spores

Oval to cylindrical-oval, sometimes irregular in size, smooth surface. Track chains with more than 10 tracks.

VII. Nonomura diagram

R(Gy) 100 SRA sm(+)(+)(+)

Streptomyces toxytricini 85-13, NRRL 15443 is suitable for the purposes of the present invention.

The cultivation of these microorganisms in the production of lipstatin can be carried out according to different fermentation methods. They can e.g. carried out in shaking flasks or in 10 1 or 200 1 and 1000 1 fermentation containers. A certain amount of spore material or mycelium of the lipstatin-producing strain is introduced into a liquid medium containing suitable carbon and nitrogen sources and the salts necessary for growth, and grown aerobically at a temperature of 20-47°C for 1- 6 days. As a carbon source, e.g. dextrin, glucose, starch, ribose and glycerin. Suitable nitrogen sources are e.g. yeast extract, peptone or soy flour. Suitable salts are preferably ammonium, magnesium and calcium salts. The fermentation is carried out at pH 6-8.

Lipstatin's isolation takes place according to known methods and can be carried out, e.g. as follows: After completion of the fermentation, the fermentation liquid is centrifuged, after which 60-90% of the activity is present in the cell mass and the rest in the centrifuge. The cell mass can then be treated with a lower alcohol such as methanol and ethanol, and extracted with the same solvent. The centrifuge can be extracted with a suitable organic solvent, e.g. with methylene chloride or acetic acid. The material recovered from the extract contains the desired lipstatin and can be enriched and purified using chromatographic methods. Suitable methods are e.g. the multiplicative extraction with the system hexane/methanol/water (50:40:9), filtration chromatography over silica gel eluting with chloroform, column chromatography on silica gel eluting with hexane, acetic acid and mixtures thereof, chromatography on non-polar carrier material under elution with polar solvents such as methanol (Reversed-Phase-Chromatographie) and high-pressure liquid chromatography.

The examples which follow below contain detailed information regarding the cultivation of Streptomyces toxytricini 85-13 and the isolation of the lipstatin.

Tetrahydrolipstatin, the compound of formula Ib, can be prepared by hydrogenating lipstatin in the presence of a suitable catalyst. Catalysts can be used, e.g. palladium/coal, platinum oxide, palladium etc. Suitable solvents are e.g. lower alcohols such as methanol and ethanol. One preferably works at lower hydrogen pressure and at room temperature.

The compounds of formula I can be used as pharmaceuticals, e.g. in the form of pharmaceutical preparations. The pharmaceutical preparations can be administered orally, e.g. in the form of tablets, varnish tablets, dragées, hard and soft gelatin capsules, solutions, emulsions or suspensions.

When manufacturing pharmaceutical preparations, the products manufactured according to the invention can be administered with pharmaceutically inert, inorganic or organic carriers. As such carriers, for tablets, varnish tablets, dragées and hard gelatin capsules, e.g. lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof etc. are used.

For soft gelatin capsules suitable as carriers e.g. vegetable oil, wax, fat, semi-solid and liquid polyols etc.; however, depending on the nature of the active substance, no carriers are necessary in the case of soft gelatin capsules. In the preparation of solutions and syrups suitable as carriers e.g. water, polyols, sucrose, invert sugar, glucose etc.

The pharmaceutical preparations may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, dyes, flavoring agents, salts, to change the osmotic pressure buffer, coating agents or antioxidants. They may also contain other therapeutically valuable substances.

As mentioned at the outset, the compounds of formula I can be used in the fight against or prevention of diseases, in particular in the fight against or prevention of obesity and hyperlipaemia. The dosage can vary within wide limits and must of course be adapted individually in each individual case. In general, with oral administration, a dosage of approx. 0.1 mg to 100 mg/kg body weight per day be appropriate.

The compounds of formula I can also be added to industrially produced foodstuffs, in particular fat, oil, butter, margarine, chocolate and other confectionery items.

The following examples shall explain the present invention in more detail, but not limit its scope in any way.

EXAMPLE 1

a) Fermentation;

A shake flask with the pre-culture medium 3 91 is inoculated with spores

of Streptomyces toxytricini 85-13 (or vegetative mycelium thereof) and grown aerobically for 72 hours at 28°C as a shaking culture. About. 2-5 vol.% of this culture is used to inoculate a fermentation pre-culture of 10 1 with the pre-culture medium 391.

Incubate for 3 days at 28°C, blowing through

1 vvm and stirring at 400 RPM. This 10 1 pre-culture is used to inoculate a 200 1 production fermenter with the production medium N7. It is fermented for 124 hours at 28°C, blowing through 1.0 vvm and stirring at 150 RPM. Regular analyzes show after 124 hours an extracellular lipase-inhibiting activity of 53 ICj-q/itiI.

The pre-culture medium 3 91 (pH 7.0) has the following composition:

3% corn starch, 4% dextrin, 3% soy flour, 0.2% (NH^^SO^, 0.6% CaCG^ and 0.3% soybean oil. The pH value was adjusted to 17. The production medium N 7 (pH 7, 0) has the following composition: 1% potato starch, 0.5% glucose, 1% ribose, 0.5% glycerin, 0.2% peptone, 2% soy flour and 0.2% (NH4)2S04-

b) Processing:

The yeast liquid is centrifuged using a tube centrifuge,

obtaining 175 1 of culture filtrate and 12 kg of mycelium. The mycelium is discarded and the culture filtrate is heated for 10 minutes to 80°C, cooled, centrifuged once more and concentrated at 30°C in vacuum to 50 1. This concentrate is extracted with 50 1 of hexane by means of a continuously working extractor, mixing the emulsion obtained with 50 1 hexane/acetic ester (1:1) and separates the organic phase. This is dried over sodium sulphate and evaporated; 199 g of crude extract I are obtained. The aqueous phase is diluted with water to 100 1 and extracted with 100 1 of acetic acid. After evaporation of the acetic ester solution, 49 g of crude extract II are obtained.

c) Cleaning:

The crude extracts I and II are filtered in three portions over each 1 kg of silica gel 60 (0.040-0.063 mm grain size), eluting with chloroform (column: 10 x 100 cm). In this way, 18.3 g of enriched material is obtained. 178 g of this substance is filtered again, eluting with chloroform over 1 kg of silica gel. 5.29 g of active material is thus obtained, 802 mg of this substance is purified by means of reversed-phase chromatography on a commercially available Lobar ready-made column (Lichoprep RP-8, size C) under elution with methanol. 158 mg of (2S, 3S, 5S, 7Z, 10Z)-5-/"(S)-2-formamido-4-methyl-valeryloxy/-2-hexyl-3-hydroxy-7,10-hexadecadienolactone is obtained (Lipstatin), which is a yellowish oil at room temperature, waxy-crystalline at low temperatures.

Microanalysis (dried for 20 hours in high vacuum at 50°C): Calculated for C^H^r^OS (491.713): C 70.84 , H 10.04, N 2.85

Found: C 70.85, H 9.97, N 2.59.

— -20 o

Optical rotation: /°^_/^ = -19.0 C (c = 1, in chloroform) .

Mass spectrum (chemical ionization with NH^ as reagent gas): peaks i.a. at m/z 509 (M+NH<*>) and 492 (M+H<+>). ;IR spectrum (film): peaks i.a. at 3318, 3012, 2928, 2558, 2745, 1823, 1740, 1673, 1521, 1382, 1370, 1250, 1191 cm"<1>. On chemical decomposition of the lipstatin and comparison of the fragments obtained with known substances, it was absolute configuration established. ;EXAMPLE 2 ;a) Fermentation: ;With a pre-culture of Streptomyces ;toxytricini 85-13 prepared according to example 1 (shake flask and then 10 1 fermentation) a 200 1 fermentation is inoculated with the production medium N 16. Produk- ; The fermentation medium N 16 corresponds to the production medium N 7 used in example 1, however, it also contains 0.1% pork fat. The fermentation was carried out during 120 hours as in example 1. After 120 hours, the intracellular lipase-inhibiting activity 71 IC^Q/ml, the extracellular 4 IC^g/ml yeast liquid. b) Preparation; ;After completion of the fermentation, the yeast liquid was heated ;for 10 minutes to 80°C, then cooled and the cell mass was separated using a tube centrifuge n 11.4 kg mycelium; the culture filtrate is discarded. The mycelium is stirred for 30 minutes in 70 1 methanol, then the resulting suspension is filtered off. The filter cake is stirred and filtered off once more with 50 1 methanol. The combined methanolic extracts are concentrated to 1.8 1. This concentrate is extracted three times with a 2 1 butyl acetate. After evaporation, 160 g of crude extract is obtained from the combined organic phases. c) Purification: This crude extract is purified by multiplicative extraction with the system hexane/methanol/water (5:4:0.9). First, the active substance is transferred from the lower phase (uP) to the upper phase (oP). 160 g of crude extract is dissolved in 41 uP and stirred in a tube vessel with 4 1 oP. After separation of oP, the oP is extracted a second time with 4 1 fresh oP. A stable emulsion is formed to which a further 4 1 uP and oP are added, after which a good phase separation is achieved. After separation of oP, the oP is extracted two more times with 8 1 fresh oP. After evaporation, the combined OP gives 90.3 g of extract. The extracted uP is discarded. Now the active substance from oP must be transferred to uP. 90.3 g of the above extract is dissolved in 4 1 oP and extracted with 4 1 uP. After the phase separation, OP is extracted three more times with fresh OP. Then the OP is rejected. The combined uP is concentrated to 0.7 1 aqueous phase and this is extracted eight times with a total of 0.2 1 acetic acid. After evaporation, 25.8 g of product is obtained. The extracted aqueous phase is discarded. The further purification of this material takes place by filtration over 1 kg of silica gel 60 (0.040-0.063 mm grain size; column 10 x 100 cm) under elution with chloroform. 64 9 mg of product is obtained, which is chromatographed on a Lobar ready column (Lichoprep RP-8, size C) eluting with methanol (reversed phase chromatography). You get 204 mg of lipstatin, which according to the thin-layer chromatogram is pure. ;EXAMPLE 3 ;One dissolves 138 mg of lipstatin in 10 ml of ethanol, mixes with ;60 mg of 5-pros. palladium/charcoal and stir at room temperature for ;3 hours in a hydrogen atmosphere (Ballon). The catalyst is then centrifuged. The hydration product is chromatographed over a short silica gel column (1x5 cm) with chloroform. 112 mg of (2S,3S,5S)-5-(S)-2-formamido-4-methyl-vaeryloxy/-2-hexyl-3-hydroxy-hexadecanoic acid lactone (tetrahydrolipstatin) is obtained as a waxy, pale yellow solid body. ;Optical rotation: ^0^7^°= -32.0°C (c = 1, in chloroform). ;Mass spectrum (chemical ionization with NH^ as reagent gas): peaks i.a. at m/z 513 (M+NH<*>); 496 (M+H<+>) and 452 (M+H<+->co2).

IR spectrum (film): peaks i.a. at 3332, 2956, 2921, 2853, 1838, 1731, 1709, 1680, 1665, 1524, 1383, 1249 and 1200 cm"<1>.

<i>H NMR spectrum (270 MHz, CDCl 3 ): 0.89 (6H); 0.97 (6H); 1.15-1.5 (27H), 1.5-1.85 (6H); 1.9-2.25 (2H); 3.24 (1H); 4.32 (1H); 4.68 (1H); 5.03 (1H); 6.43 (1H); 8.07 and 8.21 UH) ppm.

EXAMPLE 4

a) Fermentation:

A 2 L shake culture bottle with medium 391 was inoculated with spores

of an agar slant culture of Streptomyces toxytricini 85-13

and aerobically incubated for 72 hours at 28°C. Then 2 1 pre-culture is transferred into a 50 1 fermenter with production medium N 16 and incubated at 28°C for 77 hours by blowing through 0.5 vvm. This 50 1 pre-culture is used when inoculating a 1,000 1 fermenter with medium N 16. This production fermentation is carried out at 28°C and blowing through 0.5 vvm for 91 hours, achieving a Lipstatin titer of 73 ICt- g/ml intracellular and 16 IC^g/ml extracellular. The entire yeast liquid is cooled to 2°C and centrifuged

41 kg of moist biomass is obtained which can be frozen at -20°C.

b) Processing:

37 kg of mycelium is thawed at 4°C and homogenized with approx. 40 1

water in a mixer. The obtained thin-flowing suspension is mixed with 140 1 of methanol and stirred for 20 minutes. It is then filtered over a filter cloth, after which the filter cake is extracted with 140 1 methanol. The methanol extracts are concentrated at 30°C to approximately 22 1. The resulting concentrate is diluted with water to 50 1 and extracted in the stirring vessel three times with 50 1 of hexane-acetic ester (1:1). In the second and third extractions, emulsions are obtained which can be broken by adding approx. 1.4 kg or 0.5 kg table salt. The combined organic extracts are concentrated, dried over sodium sulphate and evaporated to an oily residue. 4 28 g of crude extract is obtained.

c) Cleaning:

This crude extract is filtered in four portions over 1 kg

silica gel 60 (0.040 - 0.063 mm grain size), eluting with chloroform (column: 10 x 100 cm). You get 70 g of enriched preparation which is filtered in two portions over 1 kg of silica gel 60 under elution with hexane/acetic ester (gradient 9:1 to 4:1). 4.2 g of active material is obtained, which is purified in four portions by means of reversed phase chromatography on a Lobar ready column (Lichoprep RP-8, size C) eluting with methanol. 1.77 g of lipstatin is obtained.

Claims (1)

1. Procedure for the preparation of a compound of the general formula where A is the group characterized in that one a) in the preparation of (2S, 3S, 5S, 7Z, 10Z)-5-(S)-2-formamido-4-methyl-valeryloxY7-2-hexyl-3-hydroxy -7,10-hexadeca- dienoic acid lactone of formula aerobically growing Streptomyces toxytricin 85-13 NRRL 15443 in an aqueous culture medium containing suitable carbon and nitrogen sources and inorganic salts, and separates the produced compound of formula Ia from the culture liquid, or b) in the production of (2S,3S,5S)-5-/Ts)-2-formamido-4-methyl -valeryloc.SY7-2-hexyl-3-hydroxy-hexadecanoic acid lactone with formula hydrates the compound of formula Ia.